Many techniques have been proposed for the transfer of DNA to plants such as direct DNA uptake, microinjection of pure DNA and the use of viral or plasmid vectors. The strategies for gene transfer involve the introduction of foreign DNA into cells or protoplasts followed by integration into the nuclear genome. However, eukaryotic cells, more particularly plant cells, contain distinct subcellular compartments or organelles delimited by characteristic membrane systems which perform specialized functions within the cell.
In photosynthetic leaf cells of higher plants the most conspicuous organelles are the chloroplasts, which exist in a semi-autonomous fashion within the cell, containing their own genetic system and protein synthesis machinery, but relying upon a close cooperation with the nucleo-cytoplasmic system in their development and biosynthetic activities. The chloroplast present in leaf cells is one development stage of this organelle. Proplastids, etioplasts, amyloplasts, and chromoplasts are different stages. The embodiments of this invention apply to the organelle “at large” which will be referred to as a “chloroplast”.
The most essential function of chloroplasts is the performance of the light-driven reactions of photosynthesis including fixation of carbon dioxide. However, chloroplasts carry out many other biosynthetic processes of importance to the plant cell, such as synthesis of fatty acids. In addition, the reducing power of light-activated electrons drives the reduction of nitrites (NO2−) to ammonia (NH3) in the chloroplast; this ammonia provides the plant with nitrogen required for the synthesis of amino acids compartmentalized in the chloroplast and nucleotides.
Other functions in which the chloroplast is involved are of interest to the agriculture industry. For example, many herbicides act by blocking functions which are performed within the chloroplast. Triazine derived herbicides inhibit photosynthesis by displacing a plastoquinone molecule from its binding site in the 32 kDa polypeptide of photo system II. This 32 kDa polypeptide is encoded by the chloroplast genome and synthesized in the organelle. Mutant plants resistant to triazine herbicides have been obtained; they contain a mutant 32 kDa protein in which the plastoquinone can no longer be displaced by triazine herbicides.
Other herbicides are known to block specific steps in amino acid synthesis. For example the sulfonylureas are known to inhibit acetolactate synthase which is involved in isoleucine and valine synthesis. Glyphosate inhibits the function of 5-enol-pyruvyl-3-phospho-shikimate synthase, an enzyme involved in the synthesis of aromatic amino acids. These enzymes are nuclear encoded but are translocated as precursers into the chloroplast.
Synthesis and import into the chloroplast of precursor proteins are highly energy consuming processes. It would therefore be of interest to engineer foreign genes, particularly those which have products which are functional within a chloroplast, through the chloroplast genome instead of the nuclear genome. By chloroplast is intended both mature and immature forms as well as organelles having substantially similar function in tissues other than leaf.
Relevant Literature
Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts (immature chloroplasts) has been described. Daniell and McFadden, Proc. Nat'l Acad. Sci. (USA) (1987) 84: 6349–6353. Stable transformation of chloroplasts of Chlamydomonas reinhardtii (a green alga) using bombardment of recipient cells with high-velocity tungsten microprojectiles coated with foreign DNA has been described. See, for example, Boynton et al., Science (1988) 240: 1534–1538; Blowers et al. Plant Cell (1989) 1:123–132 and Debuchy et al., EMBO J. (1989) 8: 2803–2809. The transformation technique, using tungsten microprojectiles, is described by Klein et al., Nature (London) (1987) 7:70–73. Manipulation of chloroplast genes has been described, for example, generation of chloroplast mutants, Maliga et al., Nature (1975) 255: 401–402; protoplast fusion, Belliard et al., Mol. Gen. Genet (1978) 165:231–237; organelle inactivation, Avid et al., Plant Cell Rep. (1986) 3: 227–230; and chloroplast recombination, Medgyesy et al., Proc Nat'l Acad. Sci. USA (1985) 82: 6960–6964.
Tewari and coworkers have recently mapped two replication origins in pea cpDNA by electron microscopic analysis. Both of the origins of replication, identified as displacement loops (D loops), were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts (Cheung et al., supra, Merker et. al., Mol. Cell Biol. (1985) 8:1216–1223 and Boutry et al. Nature (London) (1987) 328:340–342).
Methods for targeting foreign gene products into chloroplasts (Shrier et al., EMBO J. (1985) 4:25–32) or mitochnodria (Boutry et al., supra) have been described. See also Tomai et al. Gen. Biol. Chem. (1988) 263:15104–15109 and U.S. Pat. No. 4,940,835 for the use of transit peptides for translocating nuclear gene products into the chloroplast. Methods for directing the transport of proteins to the chloroplast are reviewed in Kenauf TIBTECH (1987) 5:40–47. Articles relating to herbicide tolerance and resistance include Shah et al. Science (1986) 233:478–481 and Mazur et al., World Biotech (1985) 2:97–108.